Journal of Pharmacognosy and Phytochemistry 2019; 8(1): 1805-1810
E-ISSN: 2278-4136
P-ISSN: 2349-8234
JPP 2019; 8(1): 1805-1810
Received: 22-11-2018
Accepted: 24-12-2018
Lalngaihawmi
Department of Plant Pathology,
Assam Agricultural University,
Jorhat, Assam, India
Ashok Bhattacharyya
Director of Research (Agri.),
Assam agricultural University,
Jorhat, Assam, India
Correspondence
Lalngaihawmi
Department of Plant Pathology,
Assam Agricultural University,
Jorhat, Assam, India
Screening of antagonistic activity and production
of volatile and non- volatile compounds by
rhizospheric microbes against Fusarium
oxysporum f. sp. cubense in vitro
Lalngaihawmi and Ashok Bhattacharyya
Abstract
A total of 54 native rhizospheric microbes were screened under in vitro for their antagonistic effect
against Fusarium oxysporum f. sp. cubense (Foc), the causal organism of Fusarium wilt of banana. The
effect of all the rhizospheric microbes collected during the present investigation significantly differed in
terms of inhibition of radial growth of Foc. After 120 hours of incubation, Trichoderma reesei (RMF-25)
was found most promising as antagonist against Foc with 71.08 per cent inhibition of radial growth
followed by T. reesei (RMF- 13) with 70.55 per cent and T. harzianum (RMF-28) with 70.15 per cent
inhibition of radial growth of Foc. The three best promising Trichoderma spp. were further screened for
their ability to produce secondary metabolites viz. volatile and non-volatile compounds. The results
revealed that all the Trichoderma spp. significantly inhibited the test pathogen by production of toxic
metabolites and diffusible non-volatile secondary metabolites. In the case of production f toxic volatile
compounds, the per cent inhibition of radial growth of Foc was observed highest by T. reesei (RMF 25)
with 40.52 per cent inhibition which is closely followed by T. reesei (RMF 13) with 40.14 per cent and T.
harzianum (RMF 28) with 39.03 per cent inhibition. All the Trichoderma spp. were also found to
produce diffusible non-volatile metabolites where T. reesei (RMF- 25) was found to be most promising
with 35.96 per cent inhibition of radial growth followed by T. reesei (RMF- 13) with 35.22 per cent and
T. harzianum (RMF- 28) with 34.72 per cent inhibition of radial growth of the test pathogen in vitro.
Keywords: Rhizospheric microbes, in vitro, Foc, Trichoderma spp
Introduction
Fusarium wilt of banana, also known as the Panama disease caused by Fusarium oxysporum f.
sp cubense (Foc), is a historically important disease of bananas worldwide (Ploetz 1990) [19].
Even in India, the disease is widespread in almost all the banana growing states, with disease
severity as high as 80-90% in some states where susceptible cultivars are grown in large areas
(Mustaffa and Thangavelu, 2011). Foc is one of around 120 formae speciales (special forms)
of F. oxysporum which cause vascular wilts of flowering plants (Gerlach and Nirenberg, 1982;
Minerdi et al., 2008) and generally considered to be one of the most destructive formae
speciales of F. oxysporum (Ploetz, 1990). Since the first epidemic occurrence of the disease in
Panama in 1950s the disease and control methods had been studied (Leong et al., 2009). Due
to the perennial nature of bananas and the polycyclic nature of the disease, effective, long-term
management of Fusarium wilt remains a challenge and requires the development of new and
alternative management strategies (Ghag et al., 2015). The use of biocontrol agents either from
the rhizosphere or endophytes has been proved to be an environmental friendly disease
management strategy in recent years (Xue et al., 2015, Deltour et al., 2017, Fu et al., 2017) [28,
4, 10]
. Bio control agents involve a bewildering array of mechanisms in achieving disease
control (Junaid, 2013). Different spp. of Trichoderma are able to secrete 40 different
secondary metabolites that may contribute to their mycoparasitism and antibiotic action
(Meena et al., 2017). Therefore, biological control of Fusarium wilt disease has become an
increasingly popular disease management consideration because of its environmental friendly
nature which offers a potential alternative to the use of chemical pesticides and the discovery
of novel mechanisms of plant protection associated with certain microorganisms (Weller et al.,
2002; Fravel et al., 2003). Thus, understanding the potential of rhizospheric microorganisms in
plant disease management, the present work has been undertaken to test the efficacy of native
rhizospheric microbes and explore their biocontrol potential against Fusarium wilt of banana
in vitro.
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Journal of Pharmacognosy and Phytochemistry
Materials and methods
The present investigation was carried out in the Department
of Plant Pathology, Assam Agricultural University, Jorhat,
Assam.
Antagonistic effect of rhizospheric microbes against Foc
Native rhizospheric microbes were isolated and purified from
different rhizosphere of banana collected from the state of
Assam, Mizoram, Nagaland and Meghalaya. Irrespective of
bacterial and fungal isolates, all the rhizospheric microbes
were screened under in vitro by dual culture method (Dennis
and Webster, 1971) against Foc, the causal organism of
Fusarium wilt of banana. Culture discs of 5mm diameter each
of the fungal rhizospheric micro-organisms and the pathogen
were worked out with the help of a sterilized cork borer from
the margin of three days old active culture and transferred into
PDA medium contained in Petri plates (90mm diameter) on
opposite sides approximately at 10mm from the periphery of
the plate while rhizospheric bacterial isolates were streaked
on the opposite side of the Petri plate (90mm) containing Foc
isolates. A control plate having only the test pathogen was
also kept for comparison. The Petri plates were then incubated
at 25 ±2 o C. The experiment combinations were placed in
Completely Randomized Design (CRD) and each
combination was replicated three (3) times. Radial growth of
the test pathogen, Foc in the presence of rhizospheric
microbes was recorded and per cent growth inhibition of the
test fungus was calculated Recording of the antagonistic
effect was done after every 24 hours up to 120 hours. The
respective inhibition was calculated after Vincent (1947) as
follows:
Growth in control - Growth in treatment
Per cent inhibition (%) =
Growth in control
Identification of rhizospheric microbes
Identification of rhzospheric microbes was carried out only
for the three best performing rhizospheric microbes form dual
culture plate technique.
Screening of rhizospheric microbes for the production of
secondary metabolites
The three best promising rhizospheric microbes were further
screened for their ability to produce toxic volatile compounds
and diffusible non-volatile compounds.
Volatile compounds production
The three rhizospheric microbes were assayed for their ability
to produce volatile compounds following the protocol given
by Dennis and Webster (1971) [5]. The rhizospheric microbes
were inoculated on the sterile Petri dishes containing PDA.
Another plate of same diameter was inoculated with actively
growing mycelia discs of Foc pathogen and then inverted over
the first plate. The junction of both the petri dishes was sealed
tightly with parafilm. PDA medium with Foc at the upper and
lower lid was maintained as control. Five replications were
maintained for each antagonist and were incubated at 25°C
for seven days. The growth of the pathogen after the
incubation was measured and per cent inhibition of mycelia
growth of the pathogen was calculated according to (Vincent,
1947) [24].
Non-volatile compounds production
For the production of non-volatile compounds by the three
effective rhizospheric microorganisms, the protocol given by
Dennis and Webster (1971) was followed. The three effective
rhizospheric microbes were placed on a sterilized cellophane
paper which was placed on top of solidified PDA media
containing Petri plates. The rhizospheric microbes were
placed separately onto the cellophane paper and incubated at
25±2°C. Control plates were also maintained with cellophane
paper only without inoculation of the test organism. Five
replications were maintained for each antagonist and were
incubated at 25°C for seven days. The plates were then
incubated for seven days to check whether the rhizospheric
microbes were able to produce diffusible non-volatile
compounds. After seven days, careful removal of the
cellophane paper along with the organism was done and the
same plates were inoculated with Foc for another five days at
25±2°C. The growth of the pathogen after the incubation was
measured and per cent inhibition of mycelial growth of the
pathogen was calculated according to (Vincent, 1947).
Statistical analysis
The data collected were subjected to statistical analysis by
fisher’s method of analysis of variance. Significance of
variance among the data were calculated out by calculating
the ‘F’ value and comparing it with tabulated value of ‘F’ at 5
percent level of probability as given by Snedecor and Cochran
(1967).
Results
Antagonistic effect of rhizospheric microbes against Foc
A total of 54 native rhizospheric microbes were screened
under in vitro to test their antagonistic effect against Foc, the
causal organism of Fusarium wilt of banana. The effect of all
the rhizospheric microbes collected during the present
investigation significantly differed in terms of inhibition of
 100
radial growth of Foc. The growth of Foc in dual culture plates
were observed to progress until they came in contact with the
leading edges of the rhizospheric microbes, after which it
ceased to grow and only the rhizospheric microbes continued
to grow. The per cent inhibition over control was calculated
every 24 hours till 120 hours of incubation. The results thus
obtained have been presented in Table 1.
At 24 hours of incubation, RMF-25 was found most
promising as antagonist against Foc with 33.33 per cent which
differed significantly from all other treatments followed by
RMF- 28 and RMF-13 with 28.84 per cent inhibition of radial
growth. The per cent inhibition recorded by the rest of the
rhizospheric microbes against Foc at 24 hours incubation
ranged from 18.58 per cent in case of RMB-2 to 28.20 per
cent in case of RMF- 3, RMF- 8 and RMF- 32.
At 48 hours of incubation, the highest inhibition was recorded
with RMF- 25 which differed significantly from all the other
treatments with 58.49 per cent inhibition followed by RMF-
13 (55.88%) and RMF- 28 (55.22%). The per cent inhibition
recorded by the rest of the rhizospheric microbes ranged from
46.07 per cent to 54.90 per cent. Per cent inhibition of radial
growth of Foc by RM- 25 in vitro significantly differed from
all other treatments. However, inhibition of radial growth of
Foc by rhizospheric microbes like RMF- 13, RMF- 28, RMF-
32, RMF- 35, RMF- 39 were found to be statistically at par.
Similar observations were recorded at 72, 96 and 120 hours of
incubation with rhizospheric microbe RMF-25 with 59.95,
67.72 and 71.08 per cent inhibition respectively. The second
highest per cent inhibition was by rhizospheric microbe RMF-
13 with 57.97, 66.45 and 70.55 per cent at 72, 96 and 12
hours respectively. The third highest per cent inhibition over
control was recorded by rhizospheric microbe RMF-28 with
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Journal of Pharmacognosy and Phytochemistry

57.49, 65.66 and 70.15 per cent at 72, 96 and 12 hours
respectively. However, at 120 hours of incubation, the
inhibition of radial growth of Foc by rhizospheric microbes
RMF- 13 and RMF- 28 were found to be statistically at par.
From the overall results of the antagonistic activities of
rhizospheric microbes against Foc, fifteen rhizospheric
microbes having the capability of inhibiting more than 65 per
cent of the test pathogen were considered to be effective
against Foc, the causal organism of Fuarium wilt of banana.
These include RMF- 3, RMF- 8, RMF- 9, RMF- 13, RMF-
14, RMF- 17, RMF- 18, RMF- 23, RMF- 24, RMF- 25, RMF-
28, RMF- 31, RMF- 32, RMF- 35 and RMF- 39. The
antagonistic effects of these promising rhizospheric microbes
have been presented in Plate 1.

Table 1. Antagonistic effect of rhizospheric microbes against Foc by dual culture technique
Growth of PI* of Foc Growth PI of Foc Growth PI of Foc Growth of PI of Foc Growth PI of Foc
Foc in presence of Foc in presence of Foc in presence Foc in presence of Foc in presence
Treatment
(cm) of RMs (cm) of RMs (cm) of RMs (cm) of RMs (cm) of RMs
24 hrs 48 hrs 72 hrs 96 hrs 120 hrs
RMF* 1+Foc 1.20 23.07 1.50 50.98 2.32 48.09 3.37 46.67 4.30 42.97
RMF 2+Foc 1.14 26.92 1.59 48.03 2.36 47.20 3.44 45.56 4.39 41.77
RMF 3+Foc 1.12 28.20 1.45 52.61 2.08 53.46 2.33 63.13 2.40 68.16
RMF 4+Foc 1.17 25.00 1.48 51.63 2.30 48.54 3.38 46.51 4.23 43.89
RMF 5+Foc 1.18 24.35 1.53 50.00 2.35 47.42 3.45 45.41 3.69 51.06
RMF 6+Foc 1.19 23.71 1.50 50.98 2.41 46.08 2.41 61.86 3.76 50.13
RMF 7+Foc 1.17 25.00 1.47 51.96 2.27 49.21 3.57 43.51 3.57 52.65
RMF 8+Foc 1.12 28.20 1.44 52.94 2.01 55.03 2.30 63.60 2.36 68.70
RMF 9+Foc 1.16 25.64 1.39 54.57 2.03 54.58 2.28 63.92 2.34 68.96
RMF 10+Foc 1.19 23.71 1.54 49.67 2.30 48.54 3.55 43.82 4.31 42.83
RMF 11+Foc 1.21 22.43 1.61 47.38 2.38 46.75 3.53 44.14 4.59 39.12
RMF 12+Foc 1.20 23.07 1.56 49.01 2.42 45.86 3.47 45.09 3.82 49.33
RMF 13+Foc 1.11 28.84 1.35 55.88 1.88 57.94 2.12 66.45 2.22 70.55
RMF 14+Foc 1.15 26.28 1.45 52.61 2.05 54.13 2.20 65.18 2.38 68.43
RMF 15+Foc 1.22 27.86 1.59 48.03 2.30 48.54 3.27 48.25 3.52 53.31
RMF 16+Foc 1.24 20.51 1.56 49.01 2.31 48.32 3.32 47.46 3.59 52.38
RMF 17+Foc 1.15 26.28 1.41 53.92 2.09 53.24 2.29 63.76 2.37 68.56
RMF 18+Foc 1.14 26.92 1.39 54.57 2.04 54.36 2.33 63.13 2.38 68.43
RMF 19+Foc 1.21 22.43 1.52 50.32 2.32 48.09 3.39 46.36 3.46 54.11
RMF 20+Foc 1.22 27.86 1.56 49.01 2.36 47.20 3.40 46.20 3.51 53.44
RMF 21+Foc 1.24 20.51 1.61 47.38 2.39 46.53 3.43 45.72 3.96 47.48
RMF 22+Foc 1.21 22.43 1.65 46.07 2.41 46.08 3.45 45.41 3.70 50.92
RMF 23+Foc 1.14 26.92 1.42 53.59 2.09 53.24 2.23 64.71 2.46 67.37
RMF 24+Foc 1.13 27.56 1.39 54.57 2.00 55.25 2.20 65.18 2.30 69.49
RMF 25+Foc 1.04 33.33 1.27 58.49 1.79 59.95 2.04 67.72 2.18 71.08
RMF 26+Foc 1.22 27.86 1.50 50.98 2.36 47.20 3.43 45.72 3.65 51.59
RMF 27+Foc 1.24 20.51 1.45 52.61 2.40 46.30 3.48 44.93 4.20 44.29
RMF 28+Foc 1.11 28.84 1.37 55.22 1.90 57.49 2.17 65.66 2.25 70.15
RMF 29+Foc 1.24 20.51 1.62 47.05 2.40 46.30 3.42 45.88 3.72 50.66
RMF 30+Foc 1.19 23.71 1.60 47.71 2.35 47.42 3.33 47.31 3.99 47.08
RMF 31+Foc 1.14 26.92 1.39 54.57 2.11 52.79 2.26 64.24 2.43 67.77
RMF 32+Foc 1.12 28.20 1.38 54.90 2.14 52.12 2.27 64.08 2.44 67.63
RMF 33+Foc 1.25 19.87 1.62 47.05 2.41 46.08 3.47 45.09 3.88 48.54
RMF 34+Foc 1.26 19.23 1.61 47.38 2.42 45.86 3.57 43.51 4.33 42.57
RMF 35+Foc 1.13 27.56 1.38 54.90 2.17 51.45 2.28 63.92 2.37 68.56
RMF 36+Foc 1.25 19.87 1.59 48.03 2.36 47.20 3.46 45.25 3.78 49.86
RMF 37+Foc 1.26 19.23 1.60 47.71 2.41 46.08 3.48 44.93 3.79 49.73
RMF 38+Foc 1.25 19.87 1.59 48.03 2.34 47.65 3.46 45.25 3.61 52.12
RMF 39+Foc 1.15 26.28 1.38 54.90 1.99 55.48 2.25 64.39 2.42 67.90
RMF 40+Foc 1.24 20.51 1.65 46.07 2.37 46.97 3.47 45.09 3.81 49.46
RMF 41+Foc 1.25 19.87 1.55 49.34 2.35 47.42 3.45 45.41 3.91 48.14
RMB* 1+Foc 1.19 23.71 1.53 50.00 2.36 47.20 3.51 44.46 4.01 46.81
RMB 2+Foc 1.27 18.58 1.58 48.36 2.37 46.97 3.53 44.14 4.22 44.03
RMB 3+Foc 1.24 20.51 1.64 46.40 2.38 46.75 3.56 43.67 3.95 47.61
RMB 4+Foc 1.25 19.87 1.65 46.07 2.35 47.42 3.58 43.35 4.19 44.42
RMB 5+Foc 1.20 23.07 1.62 47.05 2.40 46.30 3.51 44.46 4.04 46.41
RMB 6+Foc 1.21 22.43 1.66 45.75 2.41 46.08 3.54 43.98 3.93 47.87
RMB 7+Foc 1.25 19.87 1.62 47.05 2.42 45.86 3.52 44.30 3.96 47.48
RMB 8+Foc 1.23 21.15 1.63 46.73 2.39 46.53 3.55 43.82 4.07 46.02
RMB 9+Foc 1.19 23.71 1.64 46.40 2.41 46.08 3.54 43.98 4.19 44.42
RMB 10+Foc 1.20 23.07 1.64 46.40 2.42 45.86 3.57 43.51 4.14 45.09
RMB 11+Foc 1.25 19.87 1.63 46.73 2.40 46.30 3.50 44.62 4.27 43.36
RMB 12+Foc 1.26 19.23 1.64 46.40 2.42 45.86 3.57 43.51 4.37 42.02
RMB 13+Foc 1.23 21.15 1.65 46.07 2.41 46.08 3.54 43.98 4.35 42.04
Control 1.56 00.00 3.06 00.00 4.47 00.00 6.32 00.00 7.54 00.00
SE.d± 0.020 0.038 0.096 0.030 0.058
CD (p=0.05) 0.040 0.075 0.190 0.059 0.114
PI = Per cent inhibition, RMF = Rhizospheric Microbes- Fung, RMB= Rhizospheric microbes- Bacteria

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Journal of Pharmacognosy and Phytochemistry
Identification of rhizospheric microbes
The three best promising rhizospheric microbes viz. RMF-25,
RMF-13 and RMF-28 were identified by sequencing of 18s
rRNA and were identified as Trichoderma reesei (RMF-25
and RMF-13) and T. harzianum (RMF-28).
Volatile compounds production
The study on the volatile compounds production potentiality
of the three promising rhizospheric microbes revealed that
volatile compound production by the rhizospheric microbes
Table 2: Volatile compounds production test of three promising Trichoderma spp.
S. No. Rhizospheric Microbes Growth (cm) of Foc at 7 DAI
1. T. reesei (RMF-25)
2. T. reesei (RMF-13)
3. T. harzianum (RMF-28)
4. Control
SEd±
CD (p=0.05)
DAI= Days after inoculation
Non-volatile compounds production
The study on the non-volatile compounds production
potentiality of the three promising rhizospheric microbes also
revealed that non-volatile compound produced by the three
promising rhizospheric microbes were inversely proportional
the growth of Foc and directly proportional to per cent
inhibition of Foc. All the Trichoderma spp. produced non-
volatile compounds having significant effect in reducing the
radial growth of test pathogen. The results thus obtained have
Table: Non-volatile production test for three promising Trichoderma spp.
S. No. Rhizospheric Microbes Growth (cm) of Foc 7 DAI
1. T. reesei (RMF-25)
2. T. reesei (RMF-13)
3. T. harzianum (RMF-28)
4. Control
SEd±
CD (p=0.05)
DAI= Days after inoculation
Discussions
In the present investigation concerning the inhibitory effect of
native rhizospheric microbes against Fusarium wilt caused by
Fusarium oxysporum f.sp. cubens, most of the rhizospheric
microbes were reported to have antagonistic effect on the
pathogen in vitro, though at different levels. It is evident from
earlier works that rhizospheric microbes have antagonistic
effect against Foc (Akila et al., 2011; Thangavelu and Gopi,
2015; Baruah et al, 2018) [3] who reported that native
rhizospheric microbes collected from different banana
rhizosphere could inhibit the growth of Foc either singly or in
combination. Several reports have also revealed that different
species of Trichoderma possess the ability to control different
phytopathogenic diseases (Elad et al., 1998; Elad and Kapat
a) CONTROL b) RMF 3 + Foc
were inversely proportional the growth of Foc and directly
proportional to per cent inhibition of Foc. All the
Trichoderma spp. significantly inhibited the test pathogen by
production of toxic volatile compounds. The results thus
obtained have been presented in Table 2. and depicted in Plate
2. The per cent inhibition of radial growth of Foc was
observed highest by RMF 25 (40.52 %) that is closely
followed by RMF 13 (40.14 %) and RMF 28 (39.03 %).
However inhibition of radial growth of all the three
rhzizospheric microbes was found to be statistically at par.
Per cent inhibition of Foc at 7 DAI
3.20 40.52
3.22 40.14
3.28 39.03
5.38 0.00
0.15
0.06
been presented in Table 3 and depicted in Plate 3. Among the
three Trichoderma spp., RMF 25 was found most promising
in producing non-volatile compounds against Foc with 35.96
per cent inhibition of radial growth followed by RMF 13
(35.22 %) and RMF 28 (34.72 %). Per cent inhibition of
radial growth of RMF 25 significantly differed from the other
two rhizospheric microbes while RMF 13 and RMF 25 were
found to be statistically at par.
Per cent inhibition of Foc 7 DAI
5.20 35.96
5.26 35.22
5.30 34.72
8.12 0.00
0.05
0.10
1999; Xu et al., 1999; Abdel-Fattah et al., 2007, Ru and Di,
2012). The inhibition of the pathogen by Trichoderma spp.
was mainly due different modes of action of Trichoderma
spp. including production of different antifungal compounds
like volatile and non-volatile secondary metabolites (Dubey et
al., 2007, Waseem et al., 2013; Thangavelu and Gopi, 2015;
Nagamani et al., 2017) who also reported the production of
these compounds in inhibiting certain pathogens. Since the
data obtained from the present investigation also indicates
significant reduction in the growth of Foc as well as inhibition
of the pathogen by the production of volatile and non-volatile
secondary metabolites, thus it corroborates with the findings
of the earlier workers.
c) RMF 8 + Foc d) RMF 9 + Foc
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Journal of Pharmacognosy and Phytochemistry

e) RMF 13 + Foc f) RMF 14 + Foc g) RMF 17 + Foc h) RMF 18 + Foc

i) RMF 23 + Foc j) RMF 24 + Foc k) RMF 25 + Foc l) RMF 28 + Foc

m) RMF 31 + Foc n) RMF 32 + Foc o) RMF 35 + Foc p) RMF 39 + Foc

Plate 1: Antagonistic effect of fifteen effective rhizospheric microbes against Foc after 120 hours of inoculation

Plate 2. Volatile compounds production assay by promising rhizospheric microbes

Plate 3 Non-volatile compounds production assay by promising rhizospheric microbes A) B) C)

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Journal of Pharmacognosy and Phytochemistry
Acknowledgement
The authors are thankful to the advisory committee and
faculty members of the Department of Plant Pathology,
Assam Agricultural University, Jorhat, for their supportive
encouragement for the smooth completion of the work.
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